The long-term goal of this working group is to understand the cellular and molecular mechanisms that govern the development and function of the mammalian retina. The present studies are centered on the neurotrophic activity of pigment epithelium-derived factor (PEDF) on human retinoblastoma Y-79 cells as a model system. Our primary objectives are (1) to determine the molecular basis for the specific interactions of PEDF with components at the surface of the retinal cells, and (2) to characterize the structure of PEDF and its functional domains. During the past year, the work was concentrated in identifying molecules from the extracellular matrix and surfaces of retinoblastoma cells that interact with PEDF. It was found that PEDF binds to glycosaminoglycans and proteoglycans via ionic interactions. Binding of PEDF molecules to receptors in retinoblastoma cells is facilitated by a component(s) secreted by Y-79 cells into the culture media. Binding studies to retinoblastoma cells using labeled PEDF in the presence of competitors, such as excess of unlabelled PEDF; antiserum Ab-rPEDF, which blocks the neurotrophic activity of PEDF; and ovalbumin; indicated that PEDF binds specifically to retinoblastoma cells. A detergent-soluble fraction prepared from Y-79 cell plasma membranes also shared this PEDF-binding activity. Proteins in membrane detergent fractions from Y-79 cells and bovine retinae were fractionated by PEDF-affinity chromatography. A membrane protein (about 80,000-MW) with PEDF-binding properties was identified in fractions from both cell types. The full-length human and bovine PEDF were overexpressed in a eukaryotic system, namely baby hamster kidney cells. Purification and characterization indicated that both human and bovine rPEDF are produced in high levels as secreted, glycosylated, and correctly folded proteins with the same biological properties as naturally occurring PEDF. Several biophysical properties of human rPEDF, including heparin interactions, were investigated in collaboration with Dr. Peter Gettins (University of Illinois at Chicago). Bovine rPEDF mutants containing deletions about the neurotrophic region were prepared. The preparation of crystals for the determination of the structure of PEDF by x-ray crystallography is presently under way in collaboration with Drs. Craig Hyde and Timothy Mueser (NIAMS). To date, crystals of human rPEDF that diffract to low resolution have been obtained.